EP2879670A1

EP2879670A1 - Antibacterial compositions and methods

Info

Publication number: EP2879670A1
Application number: EP13826328.0A
Authority: EP
Inventors: Alena FEDAROVICH; Christopher Davies; Yuri Karl PETERSON
Original assignee: Medical University of South Carolina Foundation
Current assignee: Medical University of South Carolina Foundation
Priority date: 2012-08-01
Filing date: 2013-08-01
Publication date: 2015-06-10
Also published as: WO2014022613A1; EP2879670A4

Abstract

The invention provides for compounds and methods useful for the treatment of bacterial infection or contamination, for example in the treatment of Gram-negative infections such as those caused by, for example, Neisseria gonorrhoeae, and Gram-positive infections such as those caused by, for example, Staphylococcus aureus.

Description

ANTIBACTERIAL COMPOSITIONS AND METHODS

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Applicatioii Serial No. 61/678,427, filed August 1, 2012.

The foregoing application, and all documents cited therein ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product

specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, ail referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under NIH grant R01GM 066861- 07S1. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compounds and methods useful for the treatment of bacterial infection or contamination, for example in the treatment of Gram-negative infections such as those caused by, for example, Neisseria gonorrhoeae, and Gram-positive infections such as those caused by, for example, Staphylococcus aureus,

BACKGROUND OF THE INVENTION

Historically, β-lactams have been highly successful for the treatment of bacterial infections, but the emergence of resistance to these and other antibiotics has markedly limited the treatment options for a number of pathogens. One notable example is Neisseria gonorrhoeae, an obligate human pathogen and cause of the sexually transmitted disease gonorrhea. Gonorrhea is the second most common sexually transmitted infection in the United States, with over 350,000 cases reported in 2007 by the Centers for Disease Control (CDC). Colonizing mainly in the urogenital tract, untreated infections can lead to infertility, pelvic inflammatory disease, gonococcal arthritis in both sexes, and can increase the risk of both contracting and transmitting ! HV.

Unfortunately, N. gonorrhoeae has become the poster child for how antibiotic resistance can transform a curable contagion into a dangerous pathogen that now threatens public health. Historically, a single dose of penicil lin G was used to treat gonococcal infections, but the efficacy of this antibiotic was compromised by steadily decreasing susceptibility until its withdrawal in 1987 as a recommended treatment by the CDC. In a similar way, tetracyclines were also withdrawn and, most recently, in 2007, so were fluoroquinolones. Currently, the only recommended antibiotics to treat gonococcal infections are the extended-spectrum

cephalosporins, ceftriaxone and cefixime.

Given the history of N. gonorrhoeae, ho wever, the recent emergence of strains with intermediate resistance to cephalosporins (Ceph¹) in retrospect was inevitable. M lCs of cephalosporin for N. gonorrhoeae have now steadily increased to the point where the first cases of treatment failures with cefixime or ceftriaxone are being reported. Even more alarming, a cephalosporin-resistant (Ceph^R) strain called H041 was isolated very recently in Japan with MlCs for ceftriaxone and cefixime of 2 and 8 μ§/πύ, respectively, which is well above the breakpoints (>0.25 iig-'ml) for these antibiotics. If this trend continues, ail treatment options for gonorrhea will soon be exhausted, indicating the dire need for new classes of therapeutics.

The lethal targets for β-lactams are penicillin-binding proteins (PBPs), which are transpeptidases that catalyze the formation of peptide cross-links between adjacent glycan strands during the final stages of peptidoglycan synthesis in bacteria. Peptidoglycan envelops the bacterial cell and is essential for cell growth, division and maintenance of cell shape. PBPs share a common penicillin-binding transpeptidase (TPase) domain with an active site that contains three conserved motifs: SxxK, SxN and KTG (where x is a variable residue). The Sxx motif contains the serine nucleophile that attacks the carbon)?! carbon of the penultimate D-Ala of the peptide substrate or amide carbonyl of the β-lactam ring. PBPs recognize and react covalently with the acyl-D-Ala-D-Ala C-terminus of the peptide chains in peptidoglycan, forming an acyl-enzyme complex with a serine nucleophile in the active site and releasing the C-terminal D-AIa. The peptidyl-PBP complex then reacts with an amino group from the third residue in the peptide chain from another strand of peptidoglycan, forming a transpeptide bond and releasing the enzyme for another round of cross-linking, β-lactams such as penicillins and cephalosporins are structural analogs of the D-Ala-D-Ala terminus of peptides and inhibit PBPs by reacting eova!ently with the same serine nucleophile as the substrate, but forming a stable acyl-enzyme complex. Whilst PBPs are thus inhibited by β-lactams, normal turnover of peptidoglycan via the action of peptidoglycan hydrolases results in cel l lysis.

/V. gonorrhoeae develops resistance to β-lactams by a number of mechanisms, including alteration of the PBP targets, increased expression of the Mtr efflux pump and mutation of the porin PorBlb that restricts entry into the periplasm. The primary step in this process is the acquisition of mutated forms of PBP 2 that exhibit lowered reactivity with β-lactams and compromise the effectiveness of these agents. PBP 2 is essential for the growth ofN.

gonorrhoeae and is a validated target for β-laetam antibiotics directed against this organism, but its value as a clinical target has been diminished by mutations associated with resistance.

There is a need, therefore, for additional treatment of bacterial infection or

contamination, for example in the treatment of, inter alia, Gram-negative infections such as, for example, infection with N. gonorrhoeae .

SUMMARY OF THE IN VENTION

Provided is an antibacterial composition, comprising a therapeutically effective amou t of a penicillin-binding protein inhibitor selected from the group consisting of compounds 1 -25 or a pharmaceutically acceptable salt, hydrate or solvate thereof:

and a pharmaceutically acceptable carrier and/or excipient.

The invention additionally provides for a method for the treatment of an infection with a gram-negative or gram-positive bacteria in a mammal, comprising the step of administering a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier to said mammal in need thereof.

Further, the invention provides for a method of inhibiting growth of gram-negative or gram-positive bacteria in a patient, comprising the step of administering therapeutically effecti ve amount of a compound provided herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier to said patient in need thereof.

Accordingly, it is an object of the invention to not encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. § 1 12, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as ''comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included'', "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure l A-lH is a table of 25 potential inhibitors identified through high throughput screening. Figure 2 illustrates the fluorescence polarization of free Bocillin ¹ -FL at 0.002 - 4 μΜ.

Figure 3 illustrates the fluorescence of Bocillin ^iM-FL (1 μΜ) incubated with PBP2 and the assay window at various protein concentrations.

Figure 4 illustrates the inhibitory activity of penicillin G (0.05 - 1000 μΜ) against PBP 2 (1 μ.Μ) determined using the FP competition assay with Bociliin^, -FL (1 μΜ) and the effect of 10% DMSO on IC₅0 values. The solid line represents data for PBP without DMSO and the dashed line is PBP2 with DMSO data.

Figure 5 is a chart illustrating the statistical parameters of the FP assay determined during the initial optimization and after the high throughput screening (HTS) assay. Pc is the mean FP signal of PBP 2-bound tracer control, Nc is the mean FP signal of free Bocillin-FL, and Dc is the FP signal in the presence of 100 μΜ penicillin G. S/N (signal-to-noise), and the Z factors are as defined below in the Examples.

Figure 6 shows the results of initial HTS of 50,000 small lead compounds from

DIVERSet ChemBridge Library. Cocktails of 10 different compounds (10 combos) were tested. Each plate used for screening contained duplicates of 10X cocktails with PBP 2 and BocMin^TM-FL, 4 free Bociilin™~FL controls (Nc), 4 PBP2-bound Bocillin™-FL controls (Pc), and 4 displaced tracer controls (Dc - the FP of the Bocil lin-FL - protein at 100 μ,Μ penicil lin G, denoted by open circles). Fifty two plates with 96 10X combos each and one plate with eight 10 X combos were screened. In Figure 6, each data point is the percentage inhibition for each 10X cocktail determined using the average of two FP measurements, and the means of free Bocillin- FL (Nc) and bound Bocillin-FL (Pc) controls recorded for each corresponding plate. Data points of the displaced tracer controls (Dc - the FP of the Bocillin-FL - protein at 100 μΜ penicillin G), denoted by open circles, represent the percent of inhibition based on the average of four FP measurements. The box on the top indicates the 58 cocktails that exhibited > 80% inhibition of Bocillin-FL binding to PBP 2.

Figures 7A-7C are plots of SDS-PAGE based dose-response assays using a 0.05 - 1000 μΜ concentration range for the inhibitor with 1 μΜ PBP 2 and 10 μΜ of Bocillin-FL. Triton X- 100 (0.01%) was included in this assay to eliminate "promiscuous" inhibitors. Figure 7 A shows compound 4 which has an IC₅0 value of 50 μΜ; Figure 7B is compound 2 which has an IC₅0 of 128 μΜ; and Figure 7C is compound 7 which has an C50 of 153 μ , The data points represent the mean ± standard deviation over two replicate (compounds 4 and 2) or four replicate

(compound 7) experiments.

Figure 8 is a chart showing the ICso values, antimicrobial activities and structures of seven compounds identified by high throughput screening against PBP 2 from N. gonorrhoeae strain FA! 9. MIC values against N. gonorrhoeae strains FA19 (penicillin & cephalosporin susceptible), 35/02 (Cepii¹), and FA6140 (Pen^R) are given.

Figures 9A and 9B illustrate the docking of two compounds (9A is compound 2 and 9B is compo und 7) into the crystal structure of A⁷, gonorrhoeae PBP 2. The main chain of PBP 2 is shown as a grey ribbon, residues of the active site motifs of PBPs are shown with green bonds and additional amino acids that are predicted to form interactions with the ligand are shown in blue.

Figure 10 illustrates the similarity of compounds 4, 5, and 6 and an optimization strategy for compound 5.

Figure 1 1 illustrates pharmacophore modeling of compounds 1, 4, and 5.

DETAILED DESCRIPTION

The increasing prevalence of A', gonorrhoeae strains exhibiting resistance to third- generation cephalosporins heralds the possible demise of β-lactam antibiotics as effective treatments for gonorrhea. PBP 2 is an essential enzyme and a proven target for β-iactam antibiotics directed again st N. gonorrhoeae, b ut m utated forms of the enzyme found in resistant strains are less susceptible to inhibition by these antibiotics.

Based on the hypothesis that the utility of PBP 2 as a clinical target can be restored through inhibition of the enzyme by non- |3-Iactam compounds, high-affinity inhibitors of PBP 2 were developed, desirably to be used as anti-gonococcal agents. This approach was adopted because PBP 2 is (a) essential, (b) a proven clinical target, and (c) as a perip!asmic enzyme, it is more accessible than cytoplasmic targets. Development of compounds that will inhibit the enzyme by high affinity rather than acylation will circumvent current mechanisms of antibiotic resistance and restore PBP 2 as a clinical target.

A high throughput screening (HTS) assay was developed that uses fluorescence polarization (FP) to distinguish the presence of fluorescent penicillin, Bocillin^l '-FL, in free or PBP-bound form. This assay was used to screen a 50,000 compound library for potential inhibitors of N. gonorrhoeae PBP 2. 32 compounds were identified that exhibited greater than 50% inhibition of Boeillin-FL binding to PBP 2. These included a cephalosporin that provided validation of the assay and was excluded from further study. In addition, three compounds failed to show a concentration-dependent response and were excluded from further study. Four of the identified compounds could not be purchased but one additional analog of compound 7 was available and was added to the compounds for testing. Six out of these twenty five compounds failed concentration-dependent response in presence of Triton X-l 00. Accordingly, the antimicrobial activity of nineteen compounds was tested. Of these, eight showed antimicrobial activity against susceptible and penicillin- or eephalosporm-resisiant strains of N. gonorrhoeae. In molecular simulations using the crystal structure of PBP 2, two of these docked into the active site of the enzyme and both mediate interactions with the serine nucleophile.

This invention is based on the finding that a series of molecules targeting PBPs has antibacterial activity, as evidenced by inhibition of bacterial growth by members of that class. The compounds exhibit activity against strains of Gram-negative bacteria such as, for example, Hemophilus influenza, Klebsiella pneumonia, Ciirobacter freundii, Morganella morganii, Acinetobacter baumanii, Legionella pneumophila, Pseusomonas aeruginosa, Escherichia coli, Proteus mirabilis, Enterbacter spp,, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, Neisseria gonorrhoeae, Neisseria meningitides, Borrelia burgdorferi, syphilis, Leptospira, Moraxella catarrhalis and nonferme ting gram- negative organisms such as Burkholderia cepacia complex. The compounds with which the invention is concerned are, therefore, useful for the treatment of bacterial infection or contamination, for example in the treatment of, inter alia, Gram-negative infections.

It was also found that a series of molecules targeting PBPs has antibacterial activity against Gram-positive bacteria. Examples of Gram-positive bacteria include Staphylococcus aureus, E.faecium (and other Enterococci species such as E.faecalis, E. gallinarum, E.

casseliflavus, E. avium, and E. mundtii), S. epidermidis, S. saphrophyticus, S. haemolyticus, S. hominis, S, capitis, S. schleiferi, S. warneri, S. lugenenis, Strep pyrogenes, S, agalactiae, S. pneumoniae, A. adiacens, C. diptheriae, Lactobacillus sp., Listeria monocytogenes,

Bifidobacterium, Lactobacillus sp., Eubacterium, Acinomyces Israeli, C. botulinum, C.

perfringens, Clostridium sp, Fusobacterium, and Prevotella. The compounds with which the invention is concerned are, therefore, useful for the treatment of bacterial infection or contamination, for example in the treatment of Gram -positive infections.

While the invention is not limited by any particular hypothesis as to the mechanism of action of the compounds, it is presently believed that such activity is due, at least in pari, to the compounds inhibiting penicillin-binding proteins. To identify new compounds that inhibit PBPs by high affinity, a high throughput screening (FITS) assay was developed that uses fluorescence polarization (FP) to distinguish the presence of fluorescent penicillin, Bociilin^{i l}-FL, in free or PBP-bound form. This FP assay was used to screen a 50,000 compound library for potential inhibitors of K gonorrhoeae PBP 2. FP can distinguish free and bound Bociliin^1M-FL because, when free in solution, its tumbling results in depolarization of its emission, whereas when bound to a much larger molecule (the covalent complex with PBP 2), the emitted light maintains its initial polarization. A compound that competes with Bocillin™-FL for binding is indicated by loss of FP (AmP) at 520 nm (after excitation at 485 nm). In this assay, the compound is added to PBP 2, followed by Bociilin^-FL. and ΔηιΡ is measured and compared to the positive control (Pc) which is PBP 2-bound Bocillin^{1 !}~FL. The negative control (Nc) is free Bocillin ^!M-FL in solution and penicillin G is used as a displaced Bociliin^lM-FL control (Dc). The Z factor for this assay is 0.65, which is within the accepted range of 0.5-1.0

32 compounds were identified that exhibited greater than 50% inhibition of Bociliin^lW- FL binding to PBP 2. These included a cephalosporin that provided validation of the assay and was eliminated from further study. 3 compounds failed to show a concentration-response dose. Of the remaining 28, 4 were not commercially available. Six additional compounds failed to show a concentration-response dose in the presence of 0.01% Triton X-100 (used to eliminate promiscuous inhibitors). An analogue of compound 7 was added to the list and the antimicrobial activity of the remaining 19 compounds was tested. Of these, 8 compounds showed antimicrobial activity against susceptible and penicillin- or cephalosporin-resistant strains of N. gonorrhoeae. In molecular simulations using the crystal structure of PBP 2, two of these docked into the active site of the enzyme and both mediate interactions with the serine nucleophile.

The 25 compounds that exhibited greater than 50% inhibition of Bociliin™-FL binding to PBP 2 are illustrated in Figure 1. Numbers 1 -7 are the seven compounds which showed antimicrobial activity against penicillin-susceptible, penicillin-resistant, or cephalosporin- resistant strains of N, gonorrhoeae. The analogue of compound 7 is not included because its antimicrobial activity was not any better than its parent compound , Figure 8 is a table of these seven compounds and their testing results.

Out of these seven compounds that exhibited the lowest IC₅0 values for PBP 2 (~ 50 μΜ) and good antimicrobial activity, two (compounds 4 and 5) contain a sulfonamide group between two aromatic rings. I terestingly, a sulfonamide of similar structure was identified recently as an inhibitor of PBP2a from methicillin-resistant Staphylococcus aureus (MRSA) (Turk, S., O. Verlaine, et ai. (201 1). "New noncovalent inhibitors of penicillin-binding proteins from penicillin-resistant bacteria." PLoS One 6(5): el 9418.). Docking experiments suggested interactions between the sulfonamide carbonyl and Thr600 of the KTG motif of PBP2a.

Like compounds 4 and 5, compound 1 has two aromatic rings joined by a bridging group and showed reasonable antimicrobial activity against FA19, but less so against 6140 and 35/02. The remai ing three compounds (2, 6, and 7) that showed antimicrobial activity have different chemical structures. Compound 2 also possesses two aromatic rings, one of which contains a potentially reactive hydroxy! nitrobeiizaidehyde group. This compou d demonstrated moderate antibacterial activity against FA19, with higher MIC values against FA610 and 35/02. It was the most successful compound during the docking simulations and in this model there is an interaction with the serine nucleophile of PBP 2 (Serf 10). Compound 6 exhibited the lowest IC50 value (49 μΜ) with good MICs agamst FA19 (4 g''ml) and the two antibiotic-resistant strains (both were 8 μ§/ηι1). Compound 7 showed rather moderate enzyme inhibition (IC₅0 ~ 153 μΜ) but exhibited the highest antimicrobial activity in all N, gonorrhoeae strains. Interestingly, this compound contains a thiazolidine ring, a feature of penicillin antibiotics a d arylalkylidene iminothiazoiidin-4-ones, which inhibit some PBPs in vitro and shows antimicrobial activity. It was also successful in the docking simulations and there are predicted interactions with Ser310. The resemblance of compound 7 to a β-lactam-like compound led us to try and determine whether there is a eovalcnt interaction with PBP 2 , but no increase in mass of the protein was observed by mass spectrometry (data not shown).

Overall, there was relatively weak correlation between ICso and MIC values for the seven compounds, but this is expected for initial hits from HTS because there are a variety of factors that determine the MIC. The outer membrane of Gram-negative bacteria is a barrier to hydrophobic compounds and the lipooligosaceharide structure of V. gonorrhoeae also impacts permeability. In addition, entry of hydrophilic compounds into the periplasm is influenced by porins and the action of efflux systems can remove compounds from the periplasm. Both of the antibiotic resistant strains (FA6140 and 35/02) contain a mutation in the promoter of mtrCDE that mcreases expression of the MtrC-MtrD-MtrE efflux pump. In addition, both strams harbor an altered porB allele encoding the major outer membrane porin, which decreases influ of antibiotics into the periplasmic space. He ce, the ability of the different compounds to permeate porins or to be substrates of the efflux pump can have a profound impact on antibiotic efficacy, it is also possible that some of the compounds with low MICs are cytotoxic to N, gonorrhoeae. Further investigation of the compounds is therefore necessary to establish their in vivo mechanism of antimicrobial activity.

As used herein, the term "pharmaceutically acceptable salt" means any pharmaceutically acceptable salt of the compound of formula (I). Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including i organic a d orga ic acids and bases. Such acids include, for example, acetic, benzenes lfonic, benzoic, camphorsulfonic, citric, ethenes lfonic, dichloroacetic, formic, fumaric, gluconic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic, ma!eie, malic, mandelic, methanesulfonic, muck, nitric, oxalic, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, oxalic, p-toiuenesulfonic and the like.

Particularly preferred are fumaric, hydrochloric, hydrobromic, phosphoric, succinic, sulfuric and methanesulfonic acids. Acceptable base salts include alkali metal (e.g. sodium, potassium), alkaline earth metal (e.g. calcium, magnesium) and aluminum salts.

Useful pharmaceutical carriers for the preparation of the compositions hereof, can be solids, liquids or gases. Thus, the compositions can take the form of tablets, pills, capsules, suppositories, powders, enterically coated or other protected formulations (e.g. binding on ion- exchange resins or packaging i lipid-protein vesicles), sustained release formulations, solutions, suspensions, elixirs, aerosols, and the like. The carrier can be selected from the various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water, saline, aqueous dextrose, and glycols are preferred liquid carriers, particularly (when isotonic with the blood) for injectable solutions. For example, formulations for i travenous administration comprise sterile aqueous solutions of the active ingredients) which are prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution, and rendering the solution sterile. Suitable pharmaceutical e cipients include starch, cellulose, talc, glucose, lactose, talc, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, cthanol, and the like. The compositions may be subjected to

conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers and the like. Suitable pharmaceutical carriers and their formulation are described in Remington's Pharmaceutical Sciences by E. W. Martin. Such compositions will, in any event, contain an effective amount of the active compound together with a suitable carrier so as to prepare the proper dosage form for proper administration to the recipient.

It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. As is required in the pharmaceutical art, safe and permitted doses will be determined by clinical trial, but daily dosages can vary within wide limits and will be adjusted to the individual requirements in each particular case.

Typically, however, the dosage adopted for each route of administration when a compound is administered alone to adult humans is 0.0001 to 150 mg kg body weight. In one embodiment, the dosage is from 1 to 100 mg/kg. In another embodiment, the dosage is from 1 to 30 mg/kg. Such an amount of the active compound as determined by the attending physician or veterinarian is referred to herein, and in the claims, as a "therapeutically effective amount". Such a dosage may be given, for example, from 1 to 5 times daily. In one embodiment, such a dose may be given up to 4 times daily. For intravenous injection a suitable daily dose is from 0.0001 to 150 mg kg body weight. A daily dosage can be administered as a single dosage or according to a divided dose schedule.

In the practice of the method of the invention, a patient may be treated for up to two months with the therapeutically effective amount of the antibacterial compound. In one embodiment, treatment is from 1 to 14 days. In another embodiment, treatment is from 3 to 10 days.

In the practice of the method of the present invention, a therapeutically effective amount of any one of the compounds of this invention or a combination of any of the compounds of this invention or a pharmaceutically acceptable salt thereof, is administered via any of the usual and acceptable methods known in the art, either singly or in combination. The compounds or compositions can thus be administered, for example, ocularly, topically, orally (e.g., buccal cavity), sublingually, parenteral!)-' (e.g., intramuscularly, intravenously, or subcutaneously), rectally (e.g., by suppositories or washings), transdermally (e.g., skin electroporation) or by inhalation (e.g., by aerosol), and in the form or solid, liquid or gaseous dosages, including tablets and suspensions. The administration can be conducted in a single unit dosage form with continuous therapy or in a single dose therapy ad libitum. The therapeutic composition can also be in the form of an oil emulsion or dispersion in conjunction with a lipophilic salt such as pamoic acid, or in the form of a biodegradable sustained-release composition for subcutaneous or intramuscular administration.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

EXAMPLES

Example 1

Materials: A soluble construct of wild-type PBP 2 from the penicillin-susceptible strain ofN. gonorrhoeae FA19 was expressed and purified, as described by Powell, A. J., J, Tomberg, et ai. (2009). "Crystal structures of penicillin-binding protein 2 from penicillin-susceptible and - resistant strains of Neisseria gonorrhoeae reveal an unexpectedly subtle mechanism for antibiotic resistance," J. Biol. Chem. 284(2): 1202-1212.

Bocillin ^lM FL was obtained from Invitrogen Inc. (Carlsbad, C A). Penicillin G and γ- Globulins from bovine blood (BGG) were purchased from Sigma (St. Louis, MO). Prior to use, all reagents were diluted in an assay buffer comprising 50 mM potassium phosphate, p 8, and 0. 1 mg/mi BGG. The DIVERSet library of 50,000 small lead compounds from ChemBridge Corporation (San Diego, CA) was provided by the MUSC Drug Discovery Core (DDC). Three laboratory strains ofN. gonorrhoeae FA! 9, penici llin-resistant FA6140, and cephaiosporin- resistant Ceph' strain 35/02 were from the laboratory collection of R. Nicholas (UNC-Chapel em).

Fluorescence polarization (FP) measurements: Measurements of fluorescence and FP were performed on a Spectramax M5 microplate reader (Molecular Devices, Sunnyvale, CA) with excitation and emission wavelengths of 485 nm and 520 nm, respectively, using a 515 ran filter to block residual excitation light and to minimize background interference. Black, shallow 384-well micro plates (ProxiPlate ^s - 384 F Plus, PerkinElmer) were used to record data. To minimize the polarization effects from fluorophore that may be bound to the surface of the well, both excitation and emission data were recorded from the top of the well. Reading time was 100 ms per well. Fluorescence polarization was quantified in millipolarization units (mP) according to the equation, mP ^:::: 1000 x [(I_v - G* ) / (I_v + G^sI )], where I_v and are parallel and perpendicular emission intensity measurements, respectively, corrected for background (buffer), and G is the instrument dependent correction coefficient defined as a ratio of sensitivities of the detection system for vertically and horizontally polarized light. Using the equation G = L/Ih and SoftMax® Pro Data Acquisition & Analysis Software (Molecular Devices, Sunnyvale, CA), the G-factor was determined with dilution series of Boci llin-FL-only (free tracer). The assay window (AmP) was defined as the difference between protein-tracer sample and free-tracer, i.e. AmP = mP_s - mPfree, and is a measure of the maximum specific binding.

The mP recorded for free Bocillin-FL in the range of 0.2 - 4 μΜ was 40 - 65 mP (Figure 2). Based on the results of the steady-state binding experiments using variable concentrations of PBP 2 and Bocillin-FL, the optimal assay window (AmP) was 119 mP at a protein-Bocillin-FL ratio of 1 : 1 μΜ, At this ratio, the total fluorescence of Bocillin-FL was not significantly affected by protein binding (Figure 3). The average value of the coefficient used to correct mP measurements for the instrument bias G-factor of 1.0 ± 0.01 was determined within 0.2 - 4 uM concentration range of free Bocillin-FL and was used in subsequent FP experiments, including the ! I I S.

FP assay optimization; To calculate the G-factor, FP was measured in 10 μΐ reaction volumes for free Bocillin-FL in the concentration range 0.2, 0.5, 1, 2, 3, and 4 μΜ, where the FP signal of the fluorescent tracer was low and stable. The optimal tracer-to-protein ratio was determined in the binding experiments with increasing concentrations of PBP 2 (0.02 - 4 μ.Μ). FP was recorded after shaking the plate for 2 min followed by 30 min incubation, at which point the reaction reached its steady state (data not shown). Each experiment was performed in quadruplicate at room temperature.

To evaluate the performance of the assay, steady state concentration-response experiments were carried out using penicillin G in a competition assay with Bocillin-FL.

Penicillin G (0.05 - 1000 μΜ) was mixed with I μΜ PBP 2 and 1 μΜ Bocillin-FL, followed by a 1 hr incubation. The positive (Pc) and negative controls (Nc) were defined as the FP of the Bocillin-FL - protein and of the free tracer, respectively, in the absence of penicillin G. The FP of the Bocillin-FL - protein at 100 μΜ penicillin G was defined as a displaced tracer control (Dc). Since DMSO was used as a solvent in the compound library, the effect of 30% DMSO on the FP binding assay was also determined (Figure 4). Data points were normalized to the maximum specific binding, which defines complete saturation of PBP 2 by Bocillin-FL in the absence of penicillin G, and IC₅0 values were determined using non-linear regression analysis using GraphPad Prism version 4.00 for Windows (GraphPad Software, Inc., San Diego, CA). The IC50 values of penicillin G for PBP 2 with and without 10% DMSO were 1.0 uM (95% confidence interval 0.9 - 1.2 uM, R²=0.99) and 1.4 μΜ (95% confidence interval 1.2- 1.5 μΜ, R^' 0.9 K ). respectively.

Assay performance was assessed using the following parameters: the signal-to-noise ratio S/N ^:::: (upc - μ_ηί)/80_ηε, Ζ' and Z factors. The latter were calculated as Z' = 3 - (3SD_pc + 3SD_nc)/ (μ_ρε-μ_ηο) and Z = 1- (3SD_pc + 3SD_dc}/ (Upc-Uc), where SD_pc, SD_nc, SD_dc are standard deviations and Upc, U_flc, u_<j_c are means of recorded polarization values of Pc, Nc, and Dc, respectively. The statistical parameters, including a signai-to-noise ratio > 20, and Z - and Z- factors > 0.64, indicated the sensitivity of the FP assay and demonstrated its suitability for HTS (Figure 5).

High throughput assay and screening for the inhibitors: High throughput screening (HTS) against the ChemBridge DIVERSet library was carried out under the following conditions: 1 μΐ of each compound (10% DMSO final) in duplicate was pre-incubated with 9 μΐ of PBP 2 for 1 h at room temperature, followed by additional 30 min incubation with 2 μΐ Bocillin-FL (0.87 μΜ PBP 2 ; 1 μ\1 Bocillin-FL final). In addition to samples with the compounds, each plate also contained 2 background wells (12 μ! buffer only), and at least 4 wells each for Pc Nc, and Dc reactions. All samples used for measurements of background or controls included 10% DMSO. Initial screening was carried out with 10 mM cocktails of 10 compounds (giving a final concentration of 100 μΜ for each compound). In the initial screening of 50,000 compounds (present as cocktails of 10 compounds), 58 cocktails exhibited > 80% inhibition of Bocillin-FL binding to PBP 2 (Figure 6). Each of the 580 compounds was then tested individually and 32 of these demonstrated more than 50% inhibition. None of the "hits" were autofluorescent at 100 μΜ concentration.

Concentration-response experiments; Concentration-response experiments with the 32 compounds that demonstrated > 50%) ΔηιΡ reduction compared to maximum binding were conducted using two methods: FP-based and SDS-PAGE-based steady-state binding assays. For the FP experiments, 0.01 - 200 μΜ of each compound was incubated with PBP 2 (1 iiM) for 1 h, followed, by an additional 30 mm incubation with 1 μΜ Bocillin-FL to delect the residual activity of the labeled penicillin binding. Three compounds failed to show a dose response and were excluded from further study. A cephalosporin, which had an IC₅0 of 3 μ.Μ (the lowest of ail the "hits") was also excluded since the goal was to identify ηοη-β-lactam inhibitors. However, the successful identification of a β-lactam was validation of the effectiveness of the assay.

IC50 values for 24 of the remaining compounds (4 could not be purchased) plus an analogue of compound 7 were then measured in an SDS-PAGE concentration-response assay. PBP 2 (1 μ.Μ) in 50 mM sodium phosphate, 0.01% Triton X-100, pH 8 was incubated with 0.05 - 1000 μΜ of a compound for 1 h, followed by additional 15 min incubation with 10 μΜ

Bocillin-FL. The reaction was stopped by mixing each sample with 5 X SDS-ioading buffer, followed by boiling for 2 min. The samples were submitted to electrophoresis on 10% Mini- Protean TGX SDS-PAGE gels (Bio-Rad, Hercules, CA) to separate bound PBP 2 from free ligand. At least two independent reactions were performed in duplicate at each concentration of an inhibitor. Gels were imaged by UV scanning using a Kodak EDAS 290 imaging system (Scientific Imaging Systems Eastman Kodak, New Haven, CT, USA) and the protein bands were visualized with Coomassie stain to verify equal loading. Densitometry was performed with ImageJ 1.37v program (National Institutes of Health, USA, http://rsb.info.nih.gov/ij/). In both methods, data points were normalized to the maximum value of the fluorescence intensity, which defines complete saturation of PBP 2 by Bocillin-FL in the absence of compound, and IC₅0 values of the inhibitors were defined as the concentration of a compound at 50% of the residual activity of Bocillin-FL and calculated using GraphPad Prism. Six compounds failed to show dose response in the presence of Triton X-100. The remaining 18 compounds had 1C₅₀ values in the range of 50-900 μΜ and representative examples are shown in Figures 7A-7C.

Antimicrobial susceptibility testing: Three strains of A', gonorrhoeae were used in susceptibility tests: 1) FA19, a penicillin-and cephalosporin-susceptible strain; 2) FA6140, a penicillin-resistant but cephalosporin-susceptible strain; and 3) 35/02, a penicillin-resistant and cephalosporin intermediate-resistant strain. The disc diffusion zone method was applied for preliminary testing of "hits" identified in the screening process. For this method, 100 μΐ of a cell suspension (~1 x 10' bacteria) was added to 3 mi of GCB top agar (GC Broth containing 0.75% agar) at 50°C, which was then added to GCB plates and allowed to solidify. Five μΐ of each compound (5 ,ug) was added separately to antibiotic susceptibil ty discs (BBL), and these were pl aced on top of pl ates. Those compounds th at produced a vi sibl e zone of inhibition were th en tested for their MlCs on agar plates. Briefly, GCB agar plates containing increasing amounts of the tested compounds were spotted with 5 x 10^"' cel ls. The lowest concentration of the test compound that prevented growth of <5 colonies was defined as the MIC. These experiments were repeated three times, each producing similar results.

Nineteen compounds were tested for their ability to suppress the growth of A.

gonorrhoeae FA19. Eleven compounds did not suppress growth and were excluded (data not shown). For the remaining 8 compounds, the minimal inhibitory concentrations (MlCs) were determined against all three N, gonorrhoeae strains. Figure 8 illustrates the seven compounds and their values (one compound was an analogue of compound 7 showing similar antimicrobial activity as its parent and is therefore not shown in Figure 8). The MIC values ranged from 2 - 32 uu rni and al l excepting for compound 4 were higher in resistant strains compared to FA19. The best compound overall (7) exhibited an MIC of 2 ^ig/ml (5.6 itM) against FA 19 and 8 ₍iig/ml (22.4 μΜ) against both FA6140 and 35/02. Although there was no direct correlation between MlCs and IC50 values, compound 3 exhibited the highest IC50 and MIC values.

Table 1 , below, shows the inhibitory effect of the compounds of Figure 1 on the gram- negative bacteria Neisseria gonorrhoeae and the gram-positive bacteria Staphylococcus aureus:

Table 1 ICso (μΜ) MIC (^g/ml)

Λ&ΡΒΡ2 Ng S

(FA19)

Compound ID PAGE)

I ^'?21 4 24.7

128 4 12.9

3 898 16 18.3

4 50 16 17.5

5 56 8 16.4

6 49 4 11.1

1 153 2 4.4

8 230 ND 10.6

9 442 ND 4.6

10 109 ND >78.6

11 248 ND 20

12 182 ND >70.8

13 59 ND 70.2

14 224 ND >79.6

15 534 ND 36.3

16 144 ND 36.7

17 598 ND >67.8

18 273 ND >63.4

19 NR. ND >73.4

20 NR ND >57.4

21 NR ND - 82.4

22 NR ND >70.4

23 NR ND >46

24 NR ND 18.1

25* 210 ND 4.3

anamycin 1.2

FenlciUin G 10

NR=no concentration response

ND= not determined because disk diffusion did not show significant antimicrobial activity. *This compound is an analog of compound 7

The results indicate that the compounds are useful for treating infections from gram- positive and gram-negative bacteria.

Example 2 Molecular modeling of PBPl inhibitors: Modeling, simulations and visualizations were performed using Molecular Operating Environment (MOE) Version 2011.10 (Chemical

Computing Group Inc., Montreal, Canada). The crystal structure of wild-type /V. gonorrhoeae PBP 2 was used as the starting model for docking simulations (PDB:3EQU;as described by Powell , A. J., J. Tomberg, et al. (2009). "Crystal structures of penicillin-binding protein 2 from penicillin-susceptible and -resistant strains of Neisseria gonorrhoeae reveal an unexpectedly subtle mechanism for antibiotic resistance." l_.Biol_:..Cb ern, 284(2): 1202-1212. ), Following protonation of the main chain at pH 7.5 and addition of salt at a concentration of 0.2 M, the structure was energy mmimized using the Amber99 foreefiekl and Bom solvation model.

Ligands corresponding to the 7 compounds that displayed antimicrobial activity against /V.

gonorrhoeae were prepared in MOE, and also protonated at pH 7.5 with a salt concentration of 0.2 M. The entire protein surface was used for the simulations, in which PBP2 was held rigid and the ligand was flexed. 500 poses per ligand were derived using triangle-matching placement with London dG scoring. The top 250 poses were refined using foreefiekl placement and affinity dG scoring. The resulting top pose was then minimized again, but with both PBP 2 and the ligand allowed to flex.

The presence of a refined pose within the active site of PBP 2 in the top fraction was taken as a high probability of selective interaction. For compound 2, the number one pose out of 250 refined poses docked within the active site (Figure 9). There are three predicted contacts between this compound and PBP 2; a hydrogen bond between the ligand carbonyl and the hydroxy 1 of Ser310 (the active site nucleophiie), an ionic interaction between the amide nitrogen of Thr347 and the carboxyiic acid of compound 2, and an arene -proton interaction between the conjugated phenyl ring of compound 2 and Asn364. Compound 7 was found in the active site in the fifth pose out of 250 refined poses. In this model, hydrogen bonds are observed between Asp346 and the secondary amine of the thiazolidine ring, and between the hydroxy! of Tyr544 and the carbonyl of compound 7. Additionally, there are arene -proton interactions from the thiazolidine ring to both Thr347 and Ser362. The bromine atom appears to be totally solvated and does not interact with the protein.

Compounds 4, 5 and 6 exhibit IC50 values between 49 and 56 μΜ and provide guidance for design of a generic structure. Figure 10 shows these three compounds as wel l as

development strategy for analogs. The sulfonamide moiety of compound 5 can be replaced by isosteric and/or isoelectronic functional groups such as amides, aliphatic, and olefinic chains of varying length, secondary amines, ether, ihioether, selenium, and others. The phenolic oxygen can be replaced by a variety of groups (H, substituted amino, electron withdrawing groups (EWG), electron releasing groups (ERG), sulfhydryl, ethers). Other substitutions can be made on the aromatic rings (Ri, R₂) and the aromatic rings themselves can be replaced by isosteres such as thiophene, pyridine, pyrrole, and other ring systems.

The phannacophore derived from compounds 1 , 4, and 5 and il lustrated by Figure 11 has six features including two electron withdrawing/releasing features that represent the sulfonamide linker, three hydrophobic/aromatic features representing the conserved phenolic region and a naphthalene moiety represented by two features.

Representing the naphthalene group by two hydrophobic features, where only one feature is required to be aromatic, provides increased design flexibility and allows for greater depth in novel compound design. Typically, projection features are simple duplicatio s of the primary features, but, in the case of the PBP 2 inhibitor model, there is a unique projection feature that aligns from primary features that themselves do ot align. That is to say, although the methoxy grouped between the different compounds do not align, it is very likely they interact with the same moiety on PBP 2. By incorporating this feature into the model, there is another point of electronic interaction to serve as a basis for affinity and selectivity of the inhibitor class.

Hie invention w^riil be further described by the following numbered paragraphs:

1. An antibacterial composition, comprising a therapeutically effective amount of a penicillin-binding protein inhibitor selected from the group consisting of compounds 1-25 or a pharmaceutically acceptable salt, hydrate or solvate thereof:

and a pharmaceutically acceptable carrier and/or excipient.

2. A method for the treatment of an infection with a gram-negative bacteria in a mammal, comprising the step of administering a therapeutically effective amount of a penicil binding protein inhibitor selected from the group consisting of compounds 1 -25 or a pharmaceutically acceptable salt, hydrate or solvate thereof:

3. The method according to paragraph 2, wherein said gram-negative bacteria is N. gonorrhoeae.

4. The method according to paragraph 2, wherein said gram-negative bacteria is E. coli, Acinetobacter baumanii, Pseudomonas aeruginosa, Klebsiella pneumonia, Citrobacter freundii, Morganella morganii, Serratia marcescens and penicillin- or cephalosporin-resistant strains of N. gonorrhoeae, .

5. The method according to paragraph 2, wherein said mammal is human.

6. The method according to paragraph 2, wherein said therapeutically effective amount is 0.0001 to 150 mg/kg of body weight of said mammal.

7. The method according to paragraph 2, wherein said therapeutically effective amount is 1 to 100 mg kg of body weight of said mammal. 8. The method according to paragraph 2, wherein said therapeutically effective amount is 1 to 30 mg/kg of body weight of said mammal.

9. The method according to paragraph 2, wherem said compound is administered intravenously.

10. The method according to paragrap 2, wherein said compound is administered orally.

1 1. The method according to paragraph 2, wherein said compound is administered topically.

12. The method accordi g to paragraph 2, wherei said compound is administered by aerosol.

13. A method for the treatment of an infection with a gram-positive bacteria in a mammal, comprising the step of administering a therapeutically effective amount of a penicillin- binding protein inhibitor selected from the group consisting of compounds 1 -25 or a

pharmaceutically acceptable salt, hydrate or solvate thereof:

14. The method accordmg to paragraph 13, wherem said gram-positive bacteria is Staphylococcus aureus or Vancomycin-resistaiit Enterococci.

15. The method according to paragraph 13, wherein said mammal is human.

16. The method accordmg to paragraph 13, wherein said therapeutically effective amount is 0.0001 to 150 mg kg of body weight of said mammal.

17. The method according to paragraph 13, wherein said therapeutically effective amount is 1 to 100 mg/kg of body weight of said mammal. 18. The method according to paragraph 13, wherein said therapeutically effective amount is 1 to 30 mg kg of body weight of said mammal.

19. The method according to paragraph 13, wherem said compound is administered intravenously.

20. The method according to paragraph 13, wherein said compound is administered orally.

21. The method according to paragraph 13. wherein said compound is administered topically.

22. The method according to paragraph 13, wherem said compound is administered by aerosol.

23. A method of inhibiting growth of gram-negative or gram-positive bacteria in a patient, comprising the step of administering therapeutically effective amount of a compound penicillin-binding protein inhibitor selected from the group consisting of compounds 1 -25 or a pharmaceutically acceptable salt, hydrate or solvate thereof:

a d a pharmaceutically acceptable carrier to said patient in need thereof.

It is to be understood that the invention is not limited to the particular embodiments of the invention described above, as variations of the particular embodiments may be made and still fail within the scope of the appended claims.

Claims

CLAIMS What is claimed is:

1 . An antibacterial conrposition, comprising a therapeutically effective amount of a penici Siin-binding protein inhibitor selected from the group consisting of compounds 1 -25 or a pharmaceutically acceptable salt, hydrate or solvate thereof:

and a pharmaceutically acceptable carrier and/or excipient.

2. A method for the treatment of an infection with a gram-negative bacteria in a mammal, comprising the step of administering a therapeutically effective amount of a penicil lin- binding protein inhibitor selected from the group consisting of compounds 1-25 or a

pharmaceutically acceptable salt, hydrate or solvate thereof:

3. The method according to claim 2, wherein said gram-negative bacteria is A⁷. gonorrhoeae.

4. The method according to claim 2, wherein said gram-negative bacteria is E. coli, Acinetobacter baumanii, Pseudomonas aeruginosa, Klebsiella pneumonia, Citrobacter freundii, Morganella morganii, Serratia marcescens and penicillin- or cephalosporin-resistant strains of N. gonorrhoeae, .

5. The method according to claim 2, wherei said mammal is human.

6. The method according to claim 2, wherein said therapeutically effective amount is 0.0001 to 150 mg/kg of body weight of said mammal.

7. The method according to claim 2, wherein said therapeutically effective amount is 1 to 100 mg/kg of body weight of said mammal.

8. The method according to claim 2, wherein said therapeutically effective amount is 1 to 30 mg/kg of body weight of said mammal,

9. The method according to claim 2, wherein said compound is administered intravenously.

10. The method according to claim 2, wherein said compound is administered orally.

11. The method according to claim 2, wherein said compound is administered topically.

12. The method according to claim 2, wherein said compound is administered by aerosol.

pharmaceutically acceptable salt, hydrate or solvate thereof:

and a pharmaceuiically acceptable carrier to said mammal in need thereof.

14. The method according to claim 13, wherein said gram -positive bacteria is

Staphylococcus aureus or Vancomycin-resistant Enterococci.

15. The method accordmg to claim 13, wherem said mammal is human.

16. The method according to claim 13, wherein said therapeutjcally effective amount is 0.0001 to 150 mg_''kg of body weight of said mammal.

17. The method accordmg to claim 13, wherem said therapeutically effective amount is 1 to 100 mg/kg of body weight of said mammal .

18. The method accordi g to claim 13, wherein said therapeutically effective amoimt is 1 to 30 mg/kg of body weight of said mammal

19. The method according to claim 13, wherein said compound is administered intravenously.

20. The method accordmg to claim 13, wherein said compound is administered orally.

21. The method according to claim 13, wherein said compound is administered topically.

22. The method accordmg to claim 13, wherein said compound is administered by aerosol.

23. A method of inhibiting growth of gram-negative or gram-positive bacteria in a patient, comprising the step of administering therapeutically effective amount of a compound penicillin-binding protein inhibitor selected from the group consisting of compounds 1-25 or a pharmaceutically acceptable salt, hydrate or solvate thereof:

and a pharmaceutically acceptable carrier to said patient in need thereof.